Methods for preventing progressive tissue necrosis, reperfusion injury, bacterial translocation and adult respiratory distress syndrome

ABSTRACT

The present invention is related to a method for preventing or reducing the effects of ischemia. The ischemia may be associated with injury or reperfusion injury, such as occurs as a result of infarctions, thermal injury (burns), surgical trauma, accidental trauma, hemorrhagic shock and the like. The invention is also related to methods for preventing or reducing bacterial translocation, adult respiratory distress syndrome, adherence of blood cells and platelets to endothelial cells and pulmonary hypertension. In accordance with the present invention, these conditions are prevented or reduced by administering a dehydroepiandrosterone (DHEA) derivative as defined herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of application Ser. No.08/870,234, filed Jun. 5, 1997, now U.S. Pat. No. 5,977,095 which is acontinuation-in-part of application Ser. No. 08/580,716 filed Dec. 29,1995, now U.S. Pat. No. 5,753,640, and of application Ser. No.08/516,540, filed Aug. 18, 1995, now U.S. Pat. No. 5,846,963.Application Ser. No. 08/580,716 is in turn a continuation-in-partapplication of Ser. No. 08/516,540, Ser. No. 08/516,540 is in turn acontinuation -in-part application of Ser. No. 08/480,744, filed Jun. 7,1995, now U.S. Pat. No. 5,587,369 application Ser. No. 08/480,745, filedJun. 7, 1995, now U.S. Pat. No. 5,635,496 application Ser. No.08/480,747, filed Jun. 7, 1995, now U.S. Pat. No. 5,811,418 and ofapplication Ser. No. 08/480,748, filed Jun. 7, 1995, now U.S. Pat. No.5,686,438 Ser. No. 08/480,744 is in turn a continuation-in-part of Ser.No. 08/284,688, filed Aug. 9, 1994, now U.S. Pat. No. 5,532,230 which inturn is a continuation-in-part of Ser. No. 08/029,442, filed Mar. 11,1993, now abandoned. Each of these applications is incorporated hereinby reference.

BACKGROUND OF THE INVENTION

The present invention is related to a method for preventing or reducingthe effects of ischemia. The ischemia may be associated with injury orreperfusion injury, such as occurs as a result of infarctions, thermalinjury (burns), surgical trauma, accidental trauma, hemorrhagic shockand the like. The invention is also related to methods for preventing orreducing bacterial translocation, adult respiratory distress syndrome,adherence of blood cells and platelets to endothelial cells andpulmonary hypertension. In accordance with the present invention, theseconditions are prevented or reduced by administering adehydroepiandrosterone (DHEA) derivative.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular cases, to provideadditional details respecting the practice, are incorporated byreference, and for convenience are numerically referenced in thefollowing text and respectively grouped in the appended bibliography.

The consequences of accidental injury represent the leading causes ofdeath in the United States among young adults. The use of aggressiveresuscitation protocols has increased the chances of a patient survivingthe initial trauma event following injury. However, the development ofinfectious complications still represents a significant problem in theseindividuals. Infection and the pathologic consequences of infectioncontribute significantly to the morbidity and mortality observedpost-injury (1, 2). Post-surgical complications in particular, representa frequently studied model of the array of systemic inflammatoryaberrations observed following all types of severe traumatic injury andmajor surgery (2).

It is well known that trauma patients are predisposed tolife-threatening infections as a consequence of being immunologicallycompromised (1, 2). It is believed that the negative influences on theimmune system following severe traumatic injury are similar to theprotective mechanisms involved in less severe injury. Recently, it hasbeen established that the pathophysiology of trauma/shock injury isassociated with an alteration in intestinal motility that can affect theecology of the enteric microflora and contribute to bacterialtranslocation (3, 4). In addition, increase permeability of theintestinal capillaries facilitates infiltration of microbial toxins thatinduce a systemic inflammatory syndrome mediated by potent cytokines andother bioactive substances. One of the early indicators of the systemicinflammatory syndrome is induction of an acute phase response asmeasured by production of acute phase reactants (4, 5).

It appears that infection, leading to sepsis and multiple organ failure,remains a major hurdle to overcome in the pathophysiologic response totrauma (6, 7). Thus far, therapeutic modalities designed to eithermaintain or restore organ system homeostasis in surgical and traumapatients have only been partially successful, and for the most partdisappointing. The failure to develop effective therapeutic drugs inthis area may be due to an inadequate base of knowledge upon which paststudies were designed. A better understanding is needed of the specificcomponents of the physiologic response to traumatic and surgical injury,such as a better distinction between host-protective inflammatorymechanisms from those that are host-injurious.

A number of studies have shown that multiple alterations in immunityoccur following stress and trauma. Changes in innate host resistance toinfection (3, 4), loss of memory skin test reactions (7), alteredcytokine production (8), decreased B-cell function (9), and profounddeficits in T cell responses (10) are among the most notable.Significant monocytosis following trauma has also been observed, alongwith reduced monocyte/macrophage function and increased negativeregulatory macrophage activity. These later observations are associatedwith an increased production of immunoregulatory E series prostaglandins(11). Likewise, serum immunoglobulin and protein profiles of patientsappear to be significantly altered as a consequence of trauma (12, 13).

The existence of cytokine deficits/excesses following several distinctforms of traumatic injury have been established. These reports arerelevant because lymphokines and cytokines are necessary and importantfor the induction and regulation of almost all types of immune responses(14). Recent studies have documented the existence of altered cytokinesecretion in trauma patients, as a prolonged decrease in peripheral Tcell potential for IL-2 secretion and IL-2R expression (15). Wood et al.demonstrated a persistent reduction in IL-2 production in vitro by PBMCfrom burn patients, with even lower levels of IL-2 production by T cellsfrom burn patients suffering from systemic sepsis (10). Additionally,high levels of circulating soluble IL-2R in serum from trauma patientshave been reported (10). A depression in yIFN production has been shownto occur in burned humans (16), as well as in mice (17). A number ofinvestigators have noticed that iatrogenic procedures (surgicalmanipulations, transfusions, anesthesia) induce a marked depression inthe capacity of activated T cells to produce IL-2 (18). There have alsobeen observations of increased levels of tumor necrosis factor and IL-6following burn and mechanical trauma (2, 6). These changes persisted forup to 21 days post injury (2,6). The persistence of plasma levels ofIL-6 post-trauma appears to correlate with the severity and anunsuccessful outcome of septic episodes (6), and high levels of TNF havebeen associated with mortality (19). The cytokine, IL-6, is a potentbiologic response modifier (20, for review). High blood levels have beencorrelated to a pathologic response to a variety of stress stimuli, suchas inflammation or infection (20). IL-6 possesses a multiplicity ofeffects including induction of the acute phase response (21), ELAMexpression on endothelial cells and growth of plasma cells (20). IL-6can be produced by T cells, macrophages and fibroblasts in response toappropriate stimulation (20).

The metabolic and neuroendocrine responses to injury representcomponents of the adaptive stress response (22). Following a givenstressful event, the production of many hepatic proteins (acute phasereactants) and neuroendocrine compound is altered. These changes arebelieved to enhance survivability of the host. Changes in liver functionare marked by elevations in plasma Zn²⁺, C-reactive protein,haptoglobin, α1-antitrypsin, fibrinogen, α1-acid glycoprotein and anumber of heat-shock proteins. It is common to observe increasedproduction of ACTH, cortisol and some neurotransmitters (beta-endorphinand eukephalins) with concomitant decreases in estrogen and androgenproduction (24,25). The altered production of many of these diversesubstances can have pronounce effects. When an individual has anuneventful recovery from traumatic injury, neuroendocrine output andimmune responsiveness will eventually return to normal (23, 24). In thepatient sustaining severe injury, normal homeostasis of both theneuroendocrine and the immune systems become dysregulated for extendedperiods of time regardless of whether the patient recovers (18, 25).

Inflammatory stimuli such as thermal injury, major surgery andaccidental trauma are know to be potent inducers of the HPA axis. Theeffect of activating the HPA is to alter normal adrenal output ofsteroid hormones, because glucocorticoid (GCS) production is increasedat the expense of DHEAS synthesis and export. It has been clearlyestablished that thermal injury of mice has a profound and reproducibleeffect on T cell function and host resistance (26). Specifically, it hasbeen demonstrated that a number of T cell-derived lymphokines are eitherenhanced or repressed by the effect of thermal injury. These effectshave led to the hypothesis that the change in GCS and DHEA levels isresponsible for the alterations in innate and adaptive immune function.The mechanisms by which GCSs cause a depression of immunologicalfunction now appears to involve an interference with the function ofcertain nuclear transcription factors (27, 28). GCSs are now appreciatedto exert a negative influence on gene transcription through the abilityof GCS-receptor complexes to bind and inactivate the proto-oncogeneproduct cJun, which combined with cFos activates the AP-1 transcriptionsite (27, 28). Therefore, while the enhancement of gene transcriptioncaused by GCS results from a classical DNA-protein interaction (29),repression of the transcription rate of other genes by this same hormonemay result from specialized protein--protein (transcriptionfactors-hormone-receptor complexes) interactions (27, 28).

Dehydroepiandrosterone (DHEA), a weak androgen, serves as the primaryprecursor in the biosynthesis of both androgens and estrogens (30). DHEAhas been reported to play a mitigating role in obesity, diabetes,carcinogenesis, autoimmunity, neurological loss of memory (31-34), andthe negative effects of GCS on IL-2 production by murine T cells (35).

Recent insight into the mechanism of action of DHEA has come fromstudies of ischemia-induced reperfusion injury. The clinical term usedto describe the pathological process of wound extension is progressivedermal ischemia and it appears to represent the consequences of ahost-initiated, time-dependent reperfusion injury. We questioned whetherthe degree of progressive dermal ischemia and necrosis of the skinfollowing thermal injury in a murine model would be significantlyreduced by post-burn, systemic administration of the steroid hormoneDHEA (36).

DHEA and several related species of steroid hormones were evaluated fora capacity to either reduce or protect thermally injured mice againstreperfusion damage of the microvasculature. Subcutaneous administrationof DHEA at approximately 1-2 mg/kg/day achieved optimal protection.DHEA, as well as, 17α-hydroxy-pregnenolone, 16α-bromo-DHEA andandrostenediol were all protective, whereas treatment of burned animalswith other types of steroids, including androstenedione, 17β-estradiolor dihydrotestosterone had no protective effect. Additionally,intervention therapy with DHEA could be withheld for up to 4 hours afterburn with substantial therapeutic benefit (36, 75). It is desired toidentify additional compounds which could be used for protection ofpatients from reperfusion damage.

It has been observed that the immediate response to a burn injury is inmany ways similar to an experiment reperfusion injury in other tissues.Studies suggest that DHEA, either directly or indirectly, through itsaction on endothelium prevents damage to the microvasculature inreperfusion injury.

In another study the effect of DHEA on ischemia/reperfusion injury ofthe isolated rat cremaster muscle was evaluated. The experimentalapproach employed intravital microscopy to establish whether DHEApre-treatment of rats prior to ischemia/reperfusion of the isolatedmuscle would protect against damage to the capillaries and venules ofmicrocirculation. These studies indicated that in control animals, 6hours of ischemia followed by re-flow analysis at 90 minutes and 24hours lead to insufficient perfusion of the muscle. In DHEA pre-treatedrats, 6 hours of ischemia followed by re-flow analysis at 90 minutes, 24hours and even 4 days showed normal perfusion values in the isolatedmuscle. In addition, it was clear that the DHEA pre-treatment preventedsticking of neutrophils to endothelium. Additional studies in a globalischemic model demonstrated the protective effect of DHEA givenintravenously after resuscitation of clinically dead rats.

It has been recognized that the maintenance of vascular integrity is animportant response to injury. Complex hemostatic mechanisms ofcoagulation, platelet function and fibrinolysis exist to minimizeadverse consequences of vascular injury and to accelerate vascularrepair. Vascular endothelial and smooth muscle cells actively maintainvessel wall thromboresistance by expressing several antithromboticproperties. When perturbed or injured, vascular cells expressthrombogenic properties. The hemostatic properties of normal andperturbed vascular cells has been reviewed by Rodgers (38).

Interference with the supply of oxygenated blood to tissues is definedas ischemia. The effects of ischemia are known to be progressive, suchthat over time cellular vitality continues to deteriorate and tissuesbecome necrotic. Total persistent ischemia, with limited oxygenperfusion of tissues, results in cell death and eventually incoagulation-induced necrosis despite reperfusion with arterial blood.Ischemia is probably the most important cause of coagulative necrosis inhuman disease. A substantial body of evidence claims that a significantproportion of the injury associated with ischemia is a consequence ofthe events associated with reperfusion of ischemic tissues, hence theterm reperfusion injury. To place reperfusion injury into a clinicalperspective, there are three different degrees of cell injury, dependingon the duration of ischemia:

(1) With short periods of ischemia, reperfusion (and resupply of oxygen)completely restores the structural and functional integrity of the cell.Whatever degree of injury the cells have incurred can be completelyreversed upon reoxygenation. For example, changes in cellular membranepotential, metabolism and ultrastructure are short-lived if thecirculation is rapidly restored.

(2) With longer periods of ischemia, reperfusion is not associated withthe restoration of cell structure and function, but rather withdeterioration and death of cells. The response to reoxygenation in thiscase is rapid and intense inflammation.

(3) Lethal cell injury may develop during prolonged periods of ischemia,where reperfusion is not a factor.

The reversibility of cell injury as a consequence of ischemia isdetermined not only by the type and duration of the injury, but also bythe cell target. Neurons exhibit very high sensitivity to ischemia,whereas myocardial, pulmonary, hepatic and renal tissues areintermediate in sensitivity. Fibroblasts, epidermis and skeletal musclehave the lowest susceptibility to ischemic injury, requiring severalhours without blood supply to develop irreversible damage.

The proximity of the endothelium to circulating leukocytes makes it animportant early target for neutrophil adherence and subsequent damage tovascular and parenchymal tissue. Interaction of activated endothelialcells and neutrophils is an immediate early, and necessary, event inischemia/reperfusion injury (39, 40). The adhesive properties ofendothelium are rapidly induced by the influx of oxygenated blood. Inresponse to oxygen, endothelial cells become activated to produceseveral products, including leukotriene B4 (LTB4), platelet activatingfactor (PAF) and P-selectin. Leukotriene B4 is a potent neutrophilchemotactic agent (41, 42). Upon activation of the endothelial cells,P-selectin is rapidly translocated from intracellular organelles to theplasma membrane, where it acts to tether circulating neutrophils andstabilize them for activation by endothelial-bound PAF (plateletactivating factor), endothelium-derived cytokines and other biologicallyactive mediators (43). Thus, the physiologic interaction between theactivated endothelium and the activated neutrophil is recognized as acritical and immediate early event in reperfusion injury of organs andtissues. Other cellular and biochemical mediators of inflammation injurysuch as platelets, the complement cascade, and the coagulation systemare also important, but come into play much later in the cascade, in aprocess called coagulative necrosis. Finally, monocytes, macrophages,fibroblasts and smooth muscle cell infiltration are responsible forreconstruction and replacement of dead tissue with new, vital tissue, aprocess called wound healing.

A popular theory postulates a role for partially reduced, and thusactivated, oxygen species in initiation of membrane damage inreperfusion injury. Present evidence indicates that activated oxygen(superoxide, peroxide, hydroxyl radicals) is formed during ischemicepisodes and that reactive oxygen species injure ischemic cells. Toxicoxygen species are generated not during the period of ischemia itself,but rather on restoration of blood flow, or reperfusion. Two sources ofactivated oxygen species have been implicated as early events inreperfusion injury, those produced intracellularly by the xanthineoxidase pathway and those which can be transported to the extracellularenvironment by activated neutrophils (39, 40, 44-46).

In the xanthine oxidase-dependent pathway, purines derived from thecatabolism of ATP during the ischemic period provide substrates for theactivity of xanthine oxidase, which requires oxygen in catalyzing theformation of uric acid. Activated oxygen species are byproducts of thisreaction. The species of oxygen radicals derived from the xanthineoxidase pathway are O₂ ⁻ (superoxide with one electron) and H₂ O₂(hydrogen peroxide with two unpaired electrons). Superoxides aregenerated within the cytosol by xanthine oxidase (located in thecytosol). The superoxides are then catabolized to peroxides withinmitochondria by superoxide dismutase. The peroxides are furtherconverted to water either by glutathione peroxidase, in the cytosol, orby catalase in peroxisomes. Both glutathione peroxidase and catalasecomprise the antioxidant defense mechanism of most cells. The majorevidence for this hypothesis rests on the ability of allopurinol, aninhibitor of xanthine oxidase, to protect against reperfusion injury inexperimental models.

In the NADPH-dependent pathway, NADPH oxidase is activated to generatesuperoxides through reduction of molecular oxygen at the plasmamembrane. The superoxides are reduced to hydrogen peroxide by superoxidedismutase at the plasma membrane or within phagolysosomes. Finally,hydrogen peroxide within phagolysosomes can be reduced in the presenceof superoxides or ferrous iron to hydroxyl radicals. A third form ofoxygen metabolite is mediated by myloperoxidase in the presence ofchlorine to reduce hydrogen peroxide to hypochlorous acid.

The hydroxyl radical is an extremely reactive species. Mitochondrialmembranes offer a number of suitable substrates for attack by OH⁻radicals. The end result is irreversible damage to mitochondria,perpetuated by a massive influx of Ca²⁺ ions. Another probable cause ofcell death by hydroxyl radicals is through peroxidation of phospholipidsin the plasma membrane. Unsaturated fatty acids are highly susceptibletargets of hydroxyl radicals. By removing a hydrogen atom from fattyacids of cell membrane phospholipids, a free lipid radical is formed.These lipid radicals function like hydroxyl radicals to form other lipidperoxide radicals. The destruction of unsaturated fatty acids ofphospholipids leads to a loss in membrane fluidity and cell death. Someinvestigators believe that the effects of oxidative stress causeprogrammed cell death in a variety of cell types.

Infarctions and traumatic injury involve many tissues, includingvascular tissue. One response following traumatic injury is to shut downblood supply to the injured tissue. A purpose of this response is toprotect the patient from the entry of infectious agents into the body.The severe reduction in blood supply is a main factor leading toprogressive ischemia at the region of the traumatic injury. Withprogressive ischemia, tissue necrosis extends beyond the directlyaffected tissue to include surrounding unaffected tissue. Thisprogressive ischemia plays an important role in defining the ultimatetissue pathology observed in humans as a consequence of the traumaticinjury. For example, see Robson et al. (47).

One form of traumatic injury which has received a great deal ofattention is thermal injury or burns. The burn wound represents anon-uniform injury, and the spectrum of injury ranges from tissue whichis totally coagulated at the time of injury to tissue which is onlyminimally injured. Between these two extremes is tissue which isseriously damaged and not immediately destroyed, but which is destinedto die. The etiology of the progressive depth of necrosis has been shownto be stasis and thrombosis of blood flow in the dermal vessels, causingischemia and destruction of epithelial elements. This ischemia occursfor 24-48 hours following the thermal injury (47, 48). Many effects havebeen seen following a thermal injury, including adhesion of leukocytesto vessel walls, agglutination of red blood cells and liberation ofvasoactive and necrotizing substances (48).

It has been established that burn-associated microvascular occlusion andischemia are caused by the time dependent increase in development ofmicrothrombi in the zone of stasis, a condition which eventually leadsto a total occlusion of the arterioles and a microcirculatorystandstill. Whereas margination of erythrocytes, granulocytes andplatelets on venular walls are all apparent within the first few hoursfollowing thermal injury, the formation of platelet microthrombi(occurring approximately 24 hours after surgery) is believed to beresponsible for creating the conditions that cause complete andpermanent vascular occlusion and tissue destruction (49, 50). Theformation of platelet microthrombi appears to provide the cellular basisfor expanding the zone of complete occlusion and the ischemic necrosisthat advances into the zone of stasis following thermal injury.

Bacterial translocation is the process by which indigenous gut florapenetrate the intestinal barrier and invade sterile tissue. Included inthis process is the migration of microbial organisms to the drainingmesenteric lymph nodes, spleen, liver, blood and in some instances, thelung (51, 52). This phenomenon has been documented in humans followingthermal injury (53-55) and ischemia-reperfusion injury (56). DHEA hasbeen reported to be useful in reducing or preventing bacterialtranslocation (36, 75). It is desired to identify additional compoundswhich are useful for preventing or reducing bacterial translocation.

The evidence implicating the role of neutrophils in adult respiratorydistress syndrome (ARDS) is substantial but indirect (57). Some of thefirst suggestions that neutrophils may cause an ARDS-like picture werefound in severely neutropenic patients who were infused intravenouslywith donor neutrophils. Occasionally, within hours of neutrophilinfusion, there was an abrupt "white-out" of the lungs (by x-ray) andonset of ARDS symptoms. Numerous studies have shown that neutrophilsaccumulate in the lung during ARDS. For example, their presence has beendemonstrated histologically. During the early phases of ARDS, the numberof circulating whole blood cells transiently decreases, probably due totheir abnormal pulmonary sequestration. Some neutrophils that accumulatewithin lung capillaries leave the vascular space and migrate into theinterstitium and alveolar airspaces. In normal healthy volunteers,neutrophils account for less than 3% of the cells that can be obtainedby bronchoalveolar lavage (BAL). In patients with ARDS, the percentageof neutrophils in the lavage is markedly increased to 76-85%. Theaccumulation of neutrophils is associated with evidence of theiractivation. They demonstrate enhanced chemotaxis and generate abnormallyhigh levels of oxygen metabolites following in vitro stimulation.Elevated concentrations of neutrophil secretory products, such aslactoferrin, have been detected in the plasma of patients with ARDS.Further evidence that neutrophils actively participate in lung injurywas obtained from a clinical study of patients with mild lung injury whowere neutropenic for an unrelated reason (e.g., receiving chemotherapy).It was noted that lung impairment frequently worsened if a patient'shematological condition improved and circulating neutrophil countsrecovered to normal levels.

Although the evidence implicating neutrophils in the genesis of humanARDS is still largely indirect, data demonstrating the importance ofneutrophils in various animal models of acute lung injury is convincing.The common approach that has been used to demonstrate neutrophilindependence is to deplete the animal of circulating neutrophils andmeasure any diminution in lung injury that occurs. Although a number ofexperimental models have been used to study neutrophil dependence oflung injury, only a few have been selected for discussion herein becauseof space limitations.

One extensively studied model is the administration of endotoxin tosheep. When endotoxin is intravenously infused into sheep, a complex setof events occurs, one of which is increased permeability of thepulmonary capillary endothelium. This is manifested by an increase inthe flow of lung lymph which contains a higher-than-normal proteinconcentration. These changes indicate a reduction in the ability of thecapillary endothelium to retain plasma proteins within the vascularspace. The neutrophil dependence of the permeability injury wasestablished when it was found that neutrophil depletion of the sheepprior to endotoxin infusion protected them. Another in vitro model ofacute lung injury involves intravenous infusion of cobra venom factorinto rats, which causes complement activation followed byleukoaggregation and sequestration of neutrophils within the pulmonarymicrovasculature. Alveolar wall damage occurs, leading to interstitialand intra-alveolar edema with hemorrhage and fibrin deposition. Again,neutrophil depletion prevented the increased pulmonary capillary leak.

Isolated, perfused rabbit or rat lungs have also been used to studymechanisms of alveolar injury under circumstances that allow improvedcontrol of the variables that affect fluid flux. When neutrophils wereadded to the perfusate and then stimulated, albumin leaked from thevascular compartment into the lung interstitium and alveolar airspaces.Unstimulated neutrophils or stimulus alone (e.g., phorbol myristateacetate) failed to increase alveolar-capillary permeability.

As further proof that stimulated neutrophils can independently injurelung tissue, in vitro experiments have been performed using vascularendothelial and lung epithelial cells as targets. In some reports,neutrophils have been shown to detach endothelial cells or alveolarepithelial cells from the surface of the tissue culture dish. Obviously,if such an event were to occur in vivo, the denuded surfaces wouldpermit substantial leakage of plasma contents. Furthermore, many reportshave provided clear evidence that stimulated neutrophils are able tofacilitate lysis of cultured vascular endothelial cells and alveolarepithelial cells. DHEA has been reported to be useful in reducing orpreventing ARDS (36, 75). It is desired to identify additional compoundswhich are useful for preventing or reducing ARDS.

In the United States, chronic obstructive pulmonary disease (COPD)represents the fifth most common cause of death (58). COPD alsoconstitutes one of the most important causes of work incapacity andrestricted activity (59). COPD, along with many other pulmonarydiseases, causes pulmonary hypertension and right ventricularhypertrophy or cor pulmonale. Over 12 million patients in the UnitedStates alone have chronic bronchitis or emphysema, and approximately 3million are chronically hypoxic with PaO₂ <60 mmHg. These patientsdevelop hypoxic pulmonary vasoconstriction, and eventually, rightventricular hypertrophy (60). Once right ventricular hypertrophydevelops, the three-year mortality rate of those patients is 60% (61,62). Irrespective of the current management, morbidity and mortality ofpatients with COPD and pulmonary hypertension remain high.

One model to study pulmonary hypertension is the pulmonaryvasoconstriction induced by alveolar hypoxia. Experiments in isolatedanimal (63) and human (64) pulmonary arteries suggest thathypoxia-induced pulmonary vasoconstriction is mediated by a directeffect of hypoxia on pulmonary vascular smooth muscle cell. It has beenreported (65) that hypoxia can depolarize the pulmonary vascular smoothmuscle membrane by inducing an increase in tissue Na+ and a decrease inK+. More recently, it has been reported that hypoxia can alter themembrane potential in rat main pulmonary artery smooth muscle cell andcan stimulate Ca²⁺ influx through voltage-gated channels (66). There isstrong evidence that Ca²⁺ entry blockade can attenuate hypoxic pulmonaryvasoconstriction in isolated rat lung (67) and in patients with chronicobstructive lung disease (68). Conceivably, hypoxia may effect othermembrane transport mechanisms that are involved in Ca²⁺ influx and/orefflux. For example, Voelkel et al. (69) speculated that hypoxia mayimpair Ca²⁺ extrusion. Farrukh et al. (70) has demonstrated that cAMPand cGMP reverse hypoxic pulmonary vasoconstriction by stimulating Ca²⁺ATP-ase-dependent Ca²⁺ extrusion and/or redistribution. It is desired toidentify compounds which are useful for treating, reducing or preventingpulmonary hypertension.

DHEA is an endogenous androgenic steroid which has been shown to have amyriad of biological activities. Araneo et al. (26) has shown that theadministration of DHEA to burned mice within one hour after injuryresulted in the preservation of normal immunologic competence, includingthe normal capacity to produce T-cell-derived lymphokines, thegeneration of cellular immune responses and the ability to resist aninduced infection. Eich et al. (71, 72) describes the use of DHEA toreduce the rate of platelet aggregation and the use of DHEA orDHEA-sulfate (DHEA-S) to reduce the production of thromboxane,respectively.

Nestler et al. (73) shows that administration of DHEA was able in humanpatients to reduce body fat mass, increase muscle mass, lower LDLcholesterol levels without affecting HDL cholesterol levels, lower serumapolipoprotein B levels, and not affect tissue sensitivity to insulin.Kent (74) reported DHEA to be a "miracle drug" which may preventobesity, aging, diabetes mellitus and heart disease. DHEA was widelyprescribed as a drug treatment for many years. However, the Food andDrug Administration recently restricted its use. DHEA is readilyinterconvertible with its sulfate ester DHEA-S through the action ofintracellular sulfatases and sulfotransferases.

Daynes et al. (75) shows that administration of DHEA was useful for thereducing or preventing progressive tissue necrosis, reperfusion injury,bacterial translocation and adult respiratory distress syndrome.However, Daynes et al. (75) further shows that the administration ofDHEAS was not useful for reducing or preventing these pathologicalconditions.

Despite the above teaching of Daynes et al. (75), it has now beendiscovered that DHEAS can be used to reduce or prevent thepathophysiologic responses to the above noted pathological conditionswhen administered intravenously when necessary or orally at the dosesdescribed in detail below. It has also now been discovered thatadditional DHEA congeners can be used to reduce or prevent thepathophysiologic responses to the above noted pathological conditions.

SUMMARY OF THE INVENTION

The present invention is directed to a method for preventing or reducingreperfusion injury following ischemia, cellular damage associated withischemic episodes, such as infarction, traumatic injury or hemorrhagicshock, and thus to prevent or reduce the consequent progressive necrosisof tissue associated with such ischemia. The present invention is alsodirected to a method for preventing or reducing bacterial translocation.The present invention is further directed to a method for preventing orreducing ARDS. The present invention is also directed to a method forinhibiting the expression of p-selectin on endothelium. Finally, thepresent invention is directed to a method for preventing or reducingpulmonary hypertension. Reperfusion injury is prevented or reduced byadministering a dehydroepiandrosterone (DHEA) derivative to a patientfollowing, e.g., an infarction, traumatic injury hemorrhagic shock orthe like. Similarly, bacterial translocation is prevented or reduced ina patient by administering a DHEA derivative. ARDS is also prevented orreduced in a patient by administering a DHEA derivative. Similarly,p-selectin expression by the endothelium is prevented or reduced in apatient by administering a DHEA derivative. Similarly, pulmonaryhypertension is prevented or reduced in a patient by administering aDHEA derivative.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of the analysis of edema formation (earswelling) and resolution in the burned ears of control and DHEA-treatedmice.

FIG. 2 shows the analysis of edema formation (ear swelling) andresolution in the burned ears of control mice and mice treated withDHEA, androstenediol, 16α-bromo-DHEA or the known anti-glucocorticoidRU486.

FIG. 3A shows the capacity of DHEA to protect against most of theprogressive ischemia consequences of thermal injury to the ear.

FIG. 3B shows the capacity of androstenediol to protect against most ofthe progressive ischemia consequences of thermal injury to the ear.

FIG. 3C shows the capacity of 16α-bromo-DHEA to protect against most ofthe progressive ischemia consequences of thermal injury to the ear.

FIG. 3D shows the progressive ischemic consequences of thermal injury tothe ear when vehicle alone is administered.

FIG. 3E shows the progressive ischemic consequences of thermal injury tothe ear when androstenedione alone is administered.

FIG. 3F shows the progressive ischemic consequences of thermal injury tothe ear when RU486 alone is administered.

FIG. 4 shows the effect of treatment with DHEA on progressive ischemiawhen administered from 0-6 hours post-thermal injury.

FIG. 5A shows the number of flowing capillaries in proximity topost-capillary venule in Zone 1 during reperfusion injury.

FIG. 5B shows the number of flowing capillaries in proximity topost-capillary venule in Zone 2 during reperfusion injury.

FIG. 5C shows the number of flowing capillaries in proximity topost-capillary venule in Zone 3 during reperfusion injury.

FIG. 6A shows the number of leukocytes rolling through the lumen ofpost-capillary venules in a two-minute period.

FIG. 6B shows the number of leukocytes adhering or sticking to the lumenof post-capillary venules in a two-minute period.

FIG. 6C shows the number of leukocytes migrating across the endotheliumin a two-minute period.

FIG. 7A shows red cell velocity of venous blood post-reperfusion.

FIG. 7B shows red cell velocity of arterial blood post-reperfusion.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method for preventing or reducingreperfusion injury following ischemia, and cellular damage associatedwith ischemic episodes, such as infarction, traumatic injury orhemorrhagic shock. An example of an infarction is a myocardialinfarction. Examples of traumatic injury include thermal injury,surgery, chemical burns, blunt trauma or lacerations and the like. Bypreventing or reducing reperfusion injury following ischemia andcellular damage associated with ischemic episodes, the consequentprogressive necrosis of tissue associated with such infarction or injuryis also prevented or reduced. In accordance with the present invention,reperfusion injury or cellular damage associated with ischemic episodes,such as infarction, traumatic injury hemorrhagic shock or the like, isprevented or reduced by administering a dehydroepiandrosterone (DHEA)derivative intravenously to a patient as early as possible, preferablywithin six hours, more preferably within four hours, and most preferablywithin two hours, of the ischemia, infarction traumatic injury,hemorrhagic shock or the like.

The present invention is also directed to a method for preventing orreducing bacterial translocation. In accordance with the presentinvention, bacterial translocation is prevented or reduced in a patientby administering a DHEA derivative as described above. The DHEAderivative is administered within 24 hours of an injury in whichbacterial translocation is one of the sequelae.

The present invention is also directed to a method for preventing orreducing adult respiratory distress syndrome (ARDS). In accordance withthe present invention, ARDS is prevented or reduced in a patient byadministering a DHEA derivative as described above. The DHEA derivativecongener is administered prior to clinical symptoms of ARDS, primarilyto individuals at risk for ARDS. Alternatively, the DHEA derivative canbe administered orally to patients at risk for ARDS.

The present invention is also directed to a method for preventing orreducing pulmonary hypertension. In accordance with the presentinvention, pulmonary hypertension is prevented or reduced in a patientby administering a DHEA derivative as described above. The DHEA isderivative is administered to patients showing signs of pulmonaryhypertension within 24 hours of events which could lead to alveolarhypoxia.

Examples of a DHEA derivative, include but are not limited to, compoundshaving the general formulas I and II and their pharmaceuticallyacceptable salts ##STR1## wherein

R¹, R², R³, R⁴, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴ and R¹⁹ areindependently H, --OH, halogen, C₁₋₁₀ alkyl or C₁₋₁₀ alkoxy;

R⁵ is H, --OH, halogen, C₁₋₁₀ alkyl, C₁₋₁₀ alkoxy or OSO₂ R²⁰ ;

R¹⁵ is

(1) H, halogen, C₁₋₁₀ alkyl or C₁₋₁₀ alkoxy when R¹⁶ is --C(O)OR²¹ or

(2) H, halogen, OH or C₁₋₁₀ alkyl when R¹⁶ is H, halogen, OH or C₁₋₁₀alkyl or

(3) H, halogen, C₁₋₁₀ alkyl, C₁₋₁₀ alkenyl, C₁₋₁₀ alkynyl, formyl, C₁₋₁₀alkanoyl or epoxy when R¹⁶ is OH; or

R¹⁵ and R¹⁶ taken together are ═O;

R¹⁷ and R¹⁸ are independently

(1) H, --OH, halogen, C₁₋₁₀ alkyl or C₁₋₁₀ alkoxy when R¹⁶ is H, OH,halogen, C₁₋₁₀ alkyl or --C(O)OR²¹ or

(2) H, (C₁₋₁₀ alkyl)_(n) amino, (C₁₋₁₀ alkyl)_(n) amino-C₁₋₁₀ alkyl,C₁₋₁₀ alkoxy, hydroxy-C₁₋₁₀ alkyl, C₁₋₁₀ alkoxy-C₁₋₁₀ alkyl,(halogen)_(m) -C₁₋₁₀ alkyl, C₁₋₁₀ alkanoyl, formyl, C₁₋₁₀ carbalkoxy orC₁₋₁₀ alkanoyloxy when R¹⁵ and R¹⁶ taken together are ═O; or

R¹⁷ and R¹⁸ taken together are ═O or taken together with the carbon towhich they are attached form a 3-6 member ring containing 0 or 1 oxygenatom; or

R¹⁵ and R¹⁷ taken together with the carbons to which they are attachedform an epoxide ring;

R²⁰ is OH, pharmaceutically acceptable ester or pharmaceuticallyacceptable ether;

R²¹ is H, (halogen)_(m) -C₁₋₁₀ alkyl or C₁₋₁₀ alkyl;

n is 0, 1 or 2; and

m is 1, 2 or 3,

with the provisos that

(a) R³ is not H, OH or halogen when R¹, R², R⁴, R⁶, R⁷, R⁹, R¹⁰, R¹²,R¹³, R¹⁴, R¹⁷ and R¹⁹ are H and R⁵ is OH or C₁₋₁₀ alkoxy and R⁸ is H, OHor halogen and R¹¹ is H or OH and R¹⁸ is H, halogen or methyl and R¹⁵ isH and R¹⁶ is OH;

(b) R³ is not H, OH or halogen when R¹, R², R⁴, R⁶, R⁷, R⁹, R¹⁰, R¹²,R¹³, R¹⁴, R¹⁷ and R¹⁹ are H and R⁵ is OH or C₁₋₁₀ alkoxy and R⁸ is H, OHor halogen and R¹¹ is H or OH and R¹⁸ is H, halogen or methyl and R¹⁵and R¹⁶ taken together are ═O;

(c) R⁵ is not H, halogen, C₁₋₁₀ alkoxy or OSO₂ R²⁰ when R¹, R², R³, R⁴,R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁴ and R¹⁷ are H and R¹¹ is H, halogen,OH or C₁₋₁₀ alkoxy and R¹⁸ is H or halogen and R¹⁵ and R¹⁶ takentogether are ═O; and

(d) R⁵ is not H, halogen, C₁₋₁₀ alkoxy or OSO₂ R²⁰ when R¹, R², R³, R⁴,R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁴ and R¹⁷ are H and R¹¹ is H, halogen,OH or C₁₋₁₀ alkoxy and R¹⁸ is H or halogen and R¹⁵ is H and R¹⁶ is H, OHor halogen.

Compounds of general formulas I and II are synthesized as described inU.S. Pat. Nos. 4,898,694; 5,001,119; 5,028,631; and 5,175,154,incorporated herein by reference. The compounds represented by thegeneral formulas I and II exist is many stereoisomers and these formulasare intended to encompass the various stereoisomers. Examples ofrepresentative compounds which fall within the scope of general formulasI and II included the following:

5α-androstan-17-one;

16α-fluoro-5α-androstan-17-one;

3β-methyl-5α-androsten-17-one;

16α-fluoro-5α-androstan-17-one;

17β-bromo-5-androsten-16-one;

17β-fluoro-3β-methyl-5-androsten-16-one;

17α-fluoro-5α-androstan-16-one;

3β-hydroxy-5-androsten-17-one;

17α-methyl-5α-androstan-16-one;

16α-methyl-5-androsten-17-one;

3β,16α-dimethyl-5-androsten-17-one;

3β,17α-dimethyl-5-androsten-16-one;

16α-hydroxy-5-androsten-17-one;

16α-fluoro-16β-methyl-5-androsten-17-one;

16α-methyl-5α-androstan-17-one;

16-dimethylaminomethyl-5α-androstan-17-one;

16β-methoxy-5-androsten-17-one;

16α-fluoromethyl-5-androsten-17-one;

16-methylene-5-androsten-17-one;

16-cyclopropyl-5α-androstan-17-one;

16-cyclobutyl-5-androsten-17-one;

16-hydroxymethylene-5-androsten-17-one;

3α-bromo-16α-methoxy-5-androsten-17-one;

16-oxymethylene-5-androsten-17-one;

3β-methyl-16ξ-trifluoromethyl-5α-androstan-17-one;

16-carbomethoxy-5-androsten-17-one;

3β-methyl-16β-methoxy-5α-androstan-17-one;

3β-hydroxy-16α-dimethylamino-5-androsten-17-one;

17α-methyl-5-androsten-17β-ol;

17α-ethynyl-5α-androstan-17β-ol;

17β-formyl-5α-androstan-17β-ol;

20,21-epoxy-5α-pregnan-17α-ol;

3β-hydroxy-20,21-epoxy-5α-pregnan-17α-ol;

16α-fluoro-17α-ethenyl-5-androsten-17β-ol;

16α-hydroxy-5-androsten-17α-ol;

16α-methyl-5α-androstan-17α-ol;

16α-methyl-16β-fluoro-5α-androstan-17α-ol;

16α-methyl-16β-fluoro-3-hydroxy-5-androsten-17α-ol;

3β,16β-dimethyl-5-androsten-17β-ol;

3β,16,16-trimethyl-5 -androsten-17β-ol;

3β,16,16-trimethyl-5-androsten-17-one;

3β-hydroxy-4α-methyl-5-androsten-17α-ol;

3β-hydroxy-4α-methyl-5-androsten-17-one;

3α-hydroxy-1α-methyl-5-androsten-17-one;

3α-ethoxy-5α-androstan-17β-ol;

5α-pregnan-20-one;

3β-methyl-5α-pregnan-20-one;

16α-methyl-5-pregnen-20-one;

16α-methyl-3β-hydroxy-5-pregnen-20-one;

17α-fluoro-5-pregnen-20-one;

21-fluoro-5α-pregnan-20-one;

17α-methyl-5-pregnen-20-one;

20-acetoxy-cis-17(20)-5α-pregnene;

3α-methyl-16,17-epoxy-5-pregnen-20-one.

It is known that reperfusion injury, hemorrhagic shock, infarctions andtraumatic injury, such as myocardial infarctions, burns, major surgery,chemical burns, blunt trauma, lacerations and the like, can lead toinjury in which tissue necrosis extends beyond the directly affectedtissue to include surrounding unaffected tissue. This ischemia plays animportant role in defining the ultimate tissue pathology observed as aconsequence of traumatic injury in humans (47). It is also known thatone consequence of thermal injury is bacterial translocation. Thermalinjury, i.e., burns, is the best studied traumatic injury in whichprogressive ischemia occurs.

The loss of viable skin through the process of progressive ischemicnecrosis contributes significantly to much of the skin loss thatrequires surgical grafting following burn injury (76). A number ofanimal models have been developed which mimic very closely many aspectsof clinical burns. For example, following the administration of anexperimental full-thickness scald burn which covers >20% of the totalbody surface area to rodents (e.g. 72° C.) hot water exposure for 7seconds), the immediate tissue effects of the burn injury appear quitemoderate, compared to the extensive damage to the affected andsurrounding skin tissue which develops over the subsequent 24-72 hourperiod. Thus, it has been observed in both clinical and experimentalburns that the total amount of skin lost to a severe thermal injuryrepresents the sum of the immediate direct tissue destruction plus thelatent damage that occurs to the epidermis, dermis and inclusive skinstructures of the affected and surrounding skin areas.

Initial investigations using the dorsal skin thermal injury model inrodents led to some dramatic findings. It was discovered that scaldburn-injured mice that are treated within one hour after thermal injurywith the weakly androgenic steroid hormone, dehydroepiandrosterone(DHEA), develop and resolve their wounds in a manner quite distinct fromuntreated or sham treated thermally injured controls. By 3-4 days afterthermal injury, all control-injured animals demonstrate third and fourthdegree damage to the vast majority of skin tissue within the injurysite. Virtually all of the skin within the affected area is ultimatelylost as a consequence of progressive ischemic necrosis. The extent oftissue damage in these animals associates with a major loss in skinstructures (hair follicles, blood vessels, neurons, and sebaceousglands), an infiltration of fibroblasts, extensive wound contraction,and the formation of numerous fibrous adhesions under the affected skinarea. The DHEA-treated animals (about 2 mg/kg/day after an initialloading dose of 4 mg/kg), however, are observed to develop significantlyless pathology, with much less evidence of progressive damage to thedermis, subdermis and associated skin structures. Whilere-epithelialization is active in both the burn control and theDHEA-treated injured groups of mice, DHEA-treated mice demonstrate muchless wound contraction with notably less formation of fibrous adhesionsunderlying the wound site.

With the use of the dorsal skin injury model, it was clearlydemonstrated that DHEA treatment exerts a very positive influence onwound progression. These findings suggested that treatment of thermallyinjured animals with DHEA may influence wound healing based on afundamental capacity to prevent ischemia. Consequently, a modificationof the procedure first described by Boykin et al. (50) and Eriksson etal. (77) was developed to permit a kinetic evaluation and quantificationof progressive dermal ischemia during the immediate and later phases ofthermally-injured mouse ears. The technique employed in these studiesfacilitated a rigorous and sequential monitoring of the time-dependentprogression of tissue damage and ischemic necrosis in mouse earssubjected to a hot water scald burn (52° C. for 24 seconds), and hasbecome a valid animal model for investigating progressive ischemia ofburn-injured tissue.

The mouse ear consists of two layers of skin, cartilage, sparse musclecells and connective tissue. Organization of the ear vasculature iswell-ordered, comprised of arterioles, precapillary arterioles,post-capillary venules and venules. Employing an apparatus capable ofadministering controlled thermal injury to the entire surface area ofthe mouse ear, researchers have reported observing an immediate changein blood flow patterns. As a result of precise morphological studies onhemodynamic changes following burn injury of the mouse ear, threedistinct zones, easily separable by the degree of pathology, have beendescribed. These zones comprise the zone of complete capillaryocclusion, the zone of partial occlusion (stasis), and the zone ofcapillary hyperemia (50). By one hour after injury, the area of totalcapillary occlusion is restricted to the distal margin of the mouse ear.Located more proximally to this outermost, and immediately sensitivearea, is the zone of partial occlusion or stasis. It is this major areaof ear tissue which becomes progressively ischemic over the 24-72 hourperiod following thermal injury, and which ultimately undergoesnecrosis. Finally, the most proximal area of the affected ear is thezone of hyperemia. This area is fairly resistant to progressivepost-burn ischemia.

It has been discovered that the administration to a patient of atherapeutically effective amount of DHEA, DHEAS, a DHEA congener or aDHEA derivative as defined by general formulas I and II above in aphysiologically acceptable carrier as early as possible, preferablywithin four hours of a reperfusion injury, hemorrhagic shock, infarctionor traumatic injury, results in the prevention or the reduction ofreperfusion injury, hemorrhagic shock, infarction or traumaticinjury-associated ischemia. The prevention or reduction of the ischemiaresults in prevention or reduction of the consequent necrosis of tissueassociated with such ischemia. This reduction in ischemia results fromthe reduction of adherence of neutrophils to endothelial cells, as shownin the Examples. As a consequence of the reduced neutrophil adherence,the neutrophils do not become activated and do not produce cellularfactors which lead to platelet aggregation. It is most preferred thatthe DHEA derivative be administered within two hours of the patient'ssustaining the reperfusion, hemorrhagic shock, infarction or traumaticinjury. The DHEA derivative is administered to patients in otherpharmaceutically acceptable form and within binders, elixirs or otherpharmaceutically acceptable mixtures, or with other pharmaceuticallyacceptable carriers. The DHEA derivative is administered by intravenousinjection. Subsequent doses of a DHEA derivative can be administeredintravenously or orally. If the DHEA deriviative is administered priorto tissue injury, such as to a patient prior to surgery, the DHEAderivative can also be administered orally.

The physiological effects of DHEA in these similar yet different modelsof reperfusion injury have directed research towards an endothelial celltarget. In the reperfusion studies it is the microcirculatoryendothelium of the skin and muscle. In the hemorrhagic shock studiesdescribed in detail below, in which the focus is directed towards theprotective effects of DHEA, DHEAS, DHEA congeners or the DHEAderivatives of general formulas I and II on the pathology associatedfrom hemorrhagic shock, it is the microcirculatory endothelium of thegut. The gut endothelium plays a critical target in surgicalshock/trauma, as it carries the responsibility of maintaining gutbarrier function. Intervention with intravenous DHEA, DHEAS, a DHEAcongener or a DHEA derivative at specific times following resuscitationfrom hemorrhagic shock reduces or even prevents a pathophysiologicresponse. The Examples below demonstrate that DHEA or DHEAS interventionsignificantly reduces morbidity and mortality following a surgicalshock/trauma in mice. Similar results are obtained with the compounds ofgeneral formula I or general formula II, set forth above.

The change in steroid hormone levels evoked by abdominal surgery withhemorrhagic shock and resuscitation may contribute to thepathophysiologic response displayed during the post-operative period.Stress-induced elevations in plasma GCS levels that subsequentlyregulate circulating levels of DHEAS, may be a significant factorinvolved in the alterations of host resistance to infection.Complications caused by the transport of soluble toxins andtranslocation of opportunistic pathogens that are normal inhabitants ofthe gut are even more life-threatening because of their prevalence andready access to host tissue. Administration of DHEAS prevents or reducesmany of these pathophysiologic responses.

Pharmaceutical compositions containing a DHEA derivative as the activeingredient in intimate admixture with a pharmaceutical carrier can beprepared according to conventional pharmaceutical compoundingtechniques. The carrier may take a wide variety of forms depending onthe form of preparation desired for administration, e.g. intravenous ororal. In preparing the compositions in oral dosage form, any of theusual pharmaceutical media may be employed, such as, for example, water,glycols, oils, alcohols, flavoring agents, preservatives, coloringagents and the like in the case of oral liquid preparations (such as,for example, suspensions, elixirs and solutions); or carriers such asstarches, sugars, diluents, granulating agents, lubricants, binders,disintegrating agents and the like in the case of oral solidpreparations (such as, for example, powders, capsules and tablets). Ifdesired, tablets may be sugar-coated or enteric-coated by standardtechniques. The carrier may comprise sterile water, although otheringredients, for example, to aid solubility or for preservativepurposes, may be included. Injectable suspensions may also be prepared,in which case appropriate liquid carriers, suspending agents and thelike may be employed.

The dose of the DHEA derivative congener is based on well knownpharmaceutically acceptable principles to deliver a DHEA equivalent doseof, e.g., 1-200 mg/kg, preferably 2-50 mg/kg. Generally the dose of DHEAderivative necessary to deliver this level of DHEA dose or DHEAeqivalent dose is 1-1000 mg/kg, preferably 2-500 mg/kg, more preferably2-200 mg/kg. Alternatively, the dosage of DHEA derivative utilized willdeliver an equivalent of 10-100 mg/kg of DHEA. The dose of DHEAderivative necessary to deliver this level of DHEA dose or DHEAequivalent dose is 10-1,000 mg/kg, preferably 50-800 mg/kg, morepreferably, 100-500 mg/kg. The dose of DHEA derivative can be readilydetermined using conventional methods and will generally be in the rangeof the doses specified for DHEAS. For unprotected compounds, i.e., thosewhich can be sulfated by human sulfotransferases or sulfatases, it ispreferred to administer an excess dose to insure that sufficient activeagent is administered, especially if sulfatases are not active at thesite of tissue injury. The patient is treated with a DHEA derivative for3-30 days, preferably 7-14 days, following the infarction, hemorrhagicshock or traumatic injury.

For those patients who are at high risk for a myocardial infarction orat risk for reperfusion injury, it is possible to prevent or reduceprogressive ischemia associated with such an infarction or reperfusioninjury by administering a DHEA derivative prior to, simultaneouslyand/or following infarction, hemorrhagic shock or reperfusion injury inthe dosages described above. Intravenous treatment with a DHEAderivative following myocardial infarction is as described above. TheDHEA derivative can be administered to such a patient who demonstratesclassical signs for an imminent myocardial infarction in the same manneras described above, for treatment following such an infarction.Alternatively, the DHEA derivative congener can be administered orallyfor those patients at risk.

For those patients who are at risk of bacterial translocation, suchbacterial translocation is prevented or reduced by administering a DHEAderivative as described above in the dosages described above. Theadministration to prevent or reduce bacterial translocation continuesuntil the patient is no longer at risk for the bacterial translocation.

It has been discovered that it is critical that the DHEA derivative beadministered soon after reperfusion injury, hemorrhagic shock,infarction or traumatic injury in order to prevent or reduce anycellular damage. If the administration of these compounds occurs toolate, blood vessels will become occluded (initially with neutrophilsadhering to endothelial cells), at which point the administration ofthese compounds will be unable to prevent or reduce the ischemia. Thetime frame within which the administration should begin may be dependenton the type of reperfusion injury, infarction or traumatic injury, andcan be readily determined by appropriate animal models. However, it ispreferred that administration of DHEA derivative commence within fourhours, and most preferably within two hours of the ischemia, hemorrhagicshock, infarction or traumatic injury. The administration of DHEAderivative to prevent or reduce bacterial translocation should beginwithin 24 hours of the injury or stress-causing event. It is preferredthat administration of these compounds to prevent or reduce bacterialtranslocation begin within four hours, and most preferably within twohours.

ARDS is prevented or reduced by administering a DHEA derivative asdescribed above, in the dosage described above. The administration of aDHEA derivative to reduce the adherence of blood cells and platelets toendothelial cells by reducing the expression of p-selectin is asdescribed above as to dose and mode of administration, i.e.,intravenously or orally, depending on the timing of the administrationrelative to the need to reduce the adhesion. The administration of aDHEA derivative to prevent or reduce ARDS should begin before the onsetof clinical symptoms. Generally, a DHEA derivative will be administeredto patients at risk of ARDS. In this case, the DHEA derivative can beadministered orally as well.

Pulmonary hypertension is prevented or reduced by administering a DHEAderivative as described above, in the dosage described above. Generally,the DHEA derivative will be administered to patients at risk ofpulmonary hypertension. In this case, the DHEA derivative can beadministered orally as well.

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

EXAMPLE 1 Experimental Thermal-Injury Model

An experimental thermal injury model employing mouse ears was developedwhere temperature and exposure time were established empirically. Theconditions represented the minimal burn injury which progressed to totaltissue necrosis in the exposed ear of untreated mice by 24-72 hourspost-burn. Groups of Balb/c mice, approximately nine weeks old, weregiven an identifying mark, and then divided into control and treatedsubgroups. The thickness of the ear to be immersed in hot water wasrecorded, and then the entire ear of the anesthetized mouse was dippedinto 52° C. water for exactly 24 seconds. Each mouse was returned to itscage after an injection of either the propylene glycol vehicle (control)or 100 mg of test agent dissolved in propylene glycol. Ear swellingchanges were monitored on individual mice at pre-burn, and at varioushours after thermal injury.

EXAMPLE 2 Effect of DHEA in the Thermal-Injury Model

Groups of Balb/c mice, approximately 9 weeks old, were given anidentifying mark, and then divided into control and treated subgroups.The thickness of the ear to be immersed in hot water was recorded, andthen the entire ear of the anesthetized mouse was dipped into 52° C.water for exactly 24 seconds. Each mouse was returned to its cage afteran injection of either the propylene glycol vehicle (control) or 100 mgof DHEA agent dissolved in propylene glycol. Ear swelling changes weremonitored on individual mice at pre-burn, and at 1, 3, 6, 9, 12, 18, 24and 48 hours after thermal injury.

The results of the analysis of edema formation and resolution in theears of control and DHEA-treated mice are shown in FIG. 1. Ear swelling,as a measure of edema, reached a peak in both DHEA-treated and untreatedburned mice by six hours after injury. In the untreated group, theextent of swelling started to decline within 12 hours, and continued todecline rapidly over the subsequent 12 hour periods. Between 24 and 48hours post-burn, ear measurements had to be discontinued in theuntreated group due to the complete loss of ear tissue resulting fromthe complete micro- vascular occlusion of the original zone of stasis.The kinetic analysis of edema in untreated and DHEA-treatedthermally-injured mice showed that the events which take place duringthe first 24 hours following a burn-induced injury are critical to theviability of the thermally-injured tissue, such that the eventualpreservation of viable ear tissue at 48 hours correlates inversely withthe rate at which the swelling response recedes between the peak at sixhours and the final 48 hour time period.

In addition to the analysis of edema in untreated and DHEA-treatedthermally-injured mice, the changes in viability of the ear tissueitself were documented photographically. Injury of the ear tissue inmice given only the vehicle was extensive, with greater than 70% of theear tissue being necrotic and destroyed within 48 hours. The totalaffected area appeared to encompass both the zone of complete vascularocclusion and the original zone of stasis. This latter zone becamedamaged as a secondary consequence of thermal injury, a condition whichdefines progressive post-burn dermal ischemia. However, DHEA-treatedmice showed little injury and the preservation of burned ear tissue wasseen in a kinetic fashion. The only area of ear tissue that was markedlyaffected by, but not lost to the effects of thermal injury correspondedto only the original zone of complete vascular occlusion.

EXAMPLE 3 Effect of Various Compounds in the Thermal Injury Model

Groups of nine-week old thermally injured Balb/c mice were divided intosubgroups given either vehicle alone, DHEA, androstenediol,16β-bromo-DHEA, androstenedione or the potent anti-glucocorticoid,RU486. Individual mice received 100 mg of the indicated steroids or thevehicle alone immediately post-burn (day 0), and further 50 mg dosesevery 24 hours for the duration of the experiment. The ear swellingresponse of each individually marked mouse was recorded at the pre-burnstage, and at 12, 24 and 48 hours post-burn.

Burned ears of mice being treated therapeutically with androstenediol,DHEA, or the non-metabolizable, synthetic derivative of DHEA,16α-bromo-DHEA, each developed significant ear-swelling in response toburn injury (FIG. 2) and exhibited a slow and constant rate ofresolution of the swelling. This slow loss of edema following thermalinjury of the ear was paralleled by only minimal dermal ischemia andnecrosis in the area. The results of this study also confirmed that thedevelopment of edema within the burned ear of untreated mice peaks andthen recedes somewhat rapidly, such that between 24-48 hours post-burn asignificant amount of tissue ischemia and necrosis takes place. Thesimilar pattern of edema followed by progressive ischemic necrosis wasobserved with androstenedione-treated mice. Likewise, a similar patternof edema followed by progressive ischemic necrosis was observed in thegroup of thermally injured animals treated with RU486, indicating thatDHEA is not working solely via its anti-glucocorticoid effects.

FIGS. 3A-3C demonstrate the capacity of DHEA, androstenediol and16α-bromo-DHEA to protect against most of the ischemic consequences ofthermal injury to the ear. Mice treated with either one of these steroidhormones incur early changes in ear tissue with slight to no loss of eartissue several days after thermal injury. The affected area appears tocorrespond to the zone of complete occlusion defined by Boykin (50).Mice given the vehicle alone, androstenedione or RU486 (FIGS. 3D-3F)following thermal injury lose >70% of the exposed ear tissue over thefirst 48 hours post- injury due to progressive post-burn ischemicnecrosis. Without effective treatment, the areas of the burn-injured earwhich became necrotic corresponded to the zone of complete occlusionplus the zone of stasis. Thus, it was demonstrated that treatment ofthermally-injured mice with either DHEA, androstenediol, or16α-bromo-DHEA not only changes the natural course of the edema producedin the ear but also somehow protects the affected tissue fromprogressive damage by inhibiting the development of ischemia within thezone of stasis and the ultimate development of necrosis of this area.Similar results are obtained for the DHEA derivatives described above.

In similar experiments, it was found that 16α-hydroxy-DHEA was lessprotective, i.e., reduced the extent of, but did not totally preventprogressive ischemia, and 16α-chloro-DHEA was slightly protectiveagainst progressive ischemia.

EXAMPLE 4 Timing of Initial Administration of DHEA

An experiment was designed to determine whether intervention using DHEAmust be delivered immediately, or whether the intervention can bedelayed for up to several hours following burn injury. Mice wereanesthetized, administered a burn and then, while under anesthesia, fourmice were given vehicle alone, four mice were given 100 mg DHEA, and theremaining mice were divided into additional groups of four. All of themice in a single group would receive 100 mg DHEA either one, two, fouror six hours after thermal injury. Tissue loss by each mouse wasevaluated 72 hours after thermal injury, and the results of the scoringare presented in FIG. 4.

This Figure demonstrates that intervention using DHEA can be delayed forup to two hours with no significant difference in the protective effectsof DHEA mean grade of 1.25% 0.25 (p=<0.001). Even with a delay of fourhours before administration of DHEA, a mean score of 2.75% 0.479 wasobserved (p=<0.016). With a six-hour delay in delivery of DHEA, the meanscore in tissue loss was 4.0% 0.408 and was determined to besignificantly different from the group that received DHEA immediatelyafter thermal injury (p=<0.058). It was concluded that the events whichlead to necrosis are reversible by administration of DHEA for up toseveral hours post-thermal injury.

The above examples demonstrate that moderate-intensity thermal injury ofthe mouse ear is a reliable and reproducible model for examiningprogressive ischemic necrosis of the skin. The results indicate thatimmediate post-burn use of DHEA has a protective effect on thermalinjury-induced dermal ischemia. In addition to DHEA, several othersteroid hormones have been tested for their therapeutic value (see TableI).

                  TABLE 1                                                         ______________________________________                                        Results of Progressive                                                          Steroid Hormone Tested Ischemia Analysis                                      (100 mg/mouse) (mouse ear model)                                            ______________________________________                                        DHEAS            nonprotective                                                  DHEA protective                                                               16α-Bromo-DHEA protective                                               androstenediol protective                                                     androstenedione nonprotective                                                 RU 486 nonprotective                                                        ______________________________________                                    

Along with DHEA, androstenediol and 16α-bromo-DHEA were markedlyprotective, in that 90-100% of the ear tissue remained intact until theexperiment was terminated at two weeks, when the healing process wascomplete. 16α-Hydroxy-DHEA was less protective and 16α-chloro-DHEA wasslightly protective. However, DHEAS at the dose examined,androstenedione and RU486 were completely nonprotective, in that eardamage and tissue loss equivalent to untreated controls was evident inall animals within 48 hours after thermal injury. It has now beendiscovered that if a sufficiently high dose of DHEAS is administeredintravenously, following the traumatic injury, such that an equivalentamount of DHEA as used in this experiment is produced in the body, thenDHEAS is protective. Similar results are obtained for the DHEAderivatives described above.

The above Examples were repeated, in which 150 mg of DHEAS wasadministered intravenously in place of the DHEA. In these examples, thesame results were obtained with DHEA as with DHEAS.

EXAMPLE 5 Effect of DHEA on Reperfusion Injury

Male Sprague-Dawley rats weighing 130-170 g were randomly assigned to nopre-treatment, vehicle pre-treatment or DHEA pre-treatment (4 mg/kg).Animals were treated with vehicle or DHEA the day before and the day ofsurgery. Anesthesia was induced with intraperitoneal pentobarbital(60-70 mg/kg). The rats were placed on a heating pad, and bodytemperature (measured by rectal probe) was maintained at between 35-37°C. Detection of the cremaster muscle on its neurovascular pedicle wasperformed according to conventional techniques (78-80). Briefly, a skinincision is made from the anterior iliac spine to the tip of thescrotum. The testis with cremaster muscle intact is then dissected awayfrom the scrotum. An opening of 1 cm is made on the ventral surface ofthe cremaster, and the testis and spermatic cord are removed. Under amicroscope, the neurovascular pedicle, consisting of thepubic-epigastric arteries, vein, and genitofemoral nerve, is thencompletely isolated by dissecting to the origin of the vessels from theexternal iliac artery and vein. Finally, the front wall of the cremastermuscle sac is opened and the island cremaster muscle flap is preparedfor intravital videomicroscopy. The rat is secured on a speciallydesigned tissue bath, and the cremaster muscle flap is spread over thecoverglass in the opening at the bottom of the bath and fixed with 5-0silk sutures. It is then transilluminated from below, using a fiberoptictungsten lamp. The muscle is kept moist and covered with impermeableplastic film. The tissue bath, designed specifically for temperaturecontrol, is filled with 0.9% saline and the temperature maintained atbetween 35° C.-36° C. The microscope is equipped with a color videocamera. The video image of the microcirculation is displayed on a 19"monitor, where the final magnification is ×1800. Measurement ofmicrovascular activity is recorded after isolation of the muscle toestablish the pre-ischemia baseline. After proper positioning of clampsto completely shut down blood flow to the muscle flap, the duration ofthe ischemic period is six hours. Following removal of clamps to inducereperfusion injury, activity in the microvasculature is measured at 30,60 and 90 minutes post-reperfusion. In all experimental subjects,ischemia is followed by reflow and then by an initial period of flow ofblood through the microcirculation. This burst of circulatory activityis followed by marked reperfusion injury that induces loss of flow.

The following parameters are used to evaluate the state of the cremastermuscle microvasculatory system prior to ischemia and after reperfusion.

1) Density of Perfused Capillaries. The density of perfused capillariesin each of three flap regions (Zone 1, 2 and 3) is measured by countingthe number of flowing capillaries in proximity to the preselectedpost-capillary venule. Nine visual fields of capillaries are counted ateach postcapillary venule site, for a total of 27 fields per cremastermuscle flap. Results are shown in FIGS. 5A, 5B and 5C for Zones 1, 2 and3, respectively.

2) Leukocyte Count in Postcapillary Venules. Video scans of threepre-selected post-capillary venules are taken in proximal, middle anddistal flap regions. For each venule, the number of leukocytes rollingthrough the lumen, the number adhering to the endothelium and the numberhaving migrated across the endothelium over a two-minute period arerecorded. Results are shown in FIGS. 6A, 6B and 6C for rollers, strikersand diapedesis, respectively.

3) Red Blood Cell Velocities in A1 (First Order) and A2 (Second Order)Arterioles. Red blood cell velocities are recorded in the mainarterioles of the cremaster flap using a custom-made optical Dopplervelocimeter. Results are shown in FIGS. 7A and 7B, for velocity ofvenous and arterial blood, respectively.

A. Reperfusion Injury in Untreated and Vehicle-Treated Rats

Six rats were untreated and six rats were pre-treated with vehicle.Under conditions of six hours of ischemia and 90 minutes of reperfusion,the absolute number of rolling, sticking and transmigrated leukocytesincreased dramatically within 60 minutes of reperfusion and showed afurther increase at 90 minutes (FIGS. 6A-6C). A dramatic decrease wasobserved in the absolute number of perfused capillaries per high-poweredfield that were at both 30 and 60 minutes post-reperfusion, with acontinued decrease in numbers of flowing capillaries at 90 minutespost-reperfusion (FIGS. 5A-5C). Likewise, red cell velocites in A2-sizedvessels were significantly slower at 60 and 90 minutes post-reperfusion(FIGS. 7A and 7B).

B. Reperfusion Injury in DHEA-Treated Rats

Under conditions where rats were pre-treated with 4 mg/kg DHEA bysubcutaneous injection the day before and the day of surgery, a markedand highly significant protective effect of the therapy was measured.All three parameters exhibited values that were close to, or identicalwith normal values. Of major importance, it was noted that alltimepoints, endothelial-adherent properties were unchanged from baselinevalues. This conclusion is based on the fact that numbers of rolling,sticking and transmigrating leukocytes appeared remarkably similar tobaseline values (FIGS. 6A-6C). Red cell velocities in A2 arterioles wereslower to return to normal rates of flow, with velocities in some areasmeasuring 75% of normal at 90 minutes post-reperfusion (FIGS. 7A and7B). At the 90-minute timepoint, the number of capillaries flowing inthe microvasculature were not significantly different from the baselinevalues obtained prior to ischemia (FIGS. 5A-5C).

When DHEAS is substituted for DHEA at a dose 1.5 times that of the DHEAused, similar results are obtained. Similar results are obtained for theDHEA derivatives described above.

Without being bound by any theory of the physiological and biochemicaloperation of the DHEA congeners, it is believed that the anti-ischemiceffects of these compounds are due to their activity on the adhesion ofneutrophils to endothelial cells. Thus, these compounds are effective inpreventing or reducing ischemia which may result from other types oftissue injury, which can be modulated by affecting adhesion toendothelial cells. This inhibition of neutrophil adhesion preventsactivation of neutrophils and transmigration to the tissue side of theendothelium. Since transmigration of neutrophils is inhibited,neutrophil-induced massive damage to endothelial cells and parenchymalcells is prevented. Since neutrophil activation is prevented, productionof cellular factors (by neutrophils) which leads to platelet aggregationis also prevented. Thus, progressive tissue necrosis is prevented orreduced. In addition, the progressive ischemia of gut tissue (leading tobacterial translocation) and of the epidermis and of cardiac muscle andthe ischemia of the alveolar wall (leading to ARDS) are mediated throughsimilar mechanisms. Thus, these compounds are also effective inpreventing or reducing bacterial translocation and ARDS.

EXAMPLE 6 Effect of DHEA on Expression of P-Selectin by Platelets

Platelets were fractionated from freshly drawn blood (mature adults andelderly). Platelets were either utilized unwashed or washed. Washedplatelets were obtained by conventional procedures (81, 82). Briefly,blood was collected to a syringe containing 1 volume of anticoagulant(0.085 M sodium citrate, 0.065 M citric acid, 2% dextrose) to 7 volumesof blood. Routinely, 50 ml of blood was withdrawn, Blood samples werecentrifuged at 180 xg for 15 minutes at room temperature to sediment redand white blood cells. The upper two-thirds of the platelet-rich plasmasupernatant was carefully removed by aspiration, and the platelets werepelleted by centrifugation at 1100 xg for 10 minutes at roomtemperature. The supernatant was decanted and the platelets wereresuspended by gently mixing the sample in 2 ml of washing buffer(Tyrode's buffer without calcium, pH 6.50 at 37° C.). The plateletsuspension was then diluted to a volume equal to the original volume ofblood drawn with Tyrode's buffer, and centrifuged at 1100 xg for 10minutes at room temperature. The platelets were washed twice more bycentrifugation and resuspended in 5 ml of incubation buffer (washingbuffer adjusted to pH 7.4 at 37° C.). The platelets were counted in aNeubauer hemocytometer.

Washed and unwashed platelets were examined for the presence ofP-selectin by direct immunostaining. Platelets (1×10⁶) were incubatedwith phycoerythrin-conjugated either negative control antibody oranti-human P-selectin monoclonal antibody (CD62 antibody, CAMFolio,Becton-Dickinson) for 15 minutes on ice. After that time, samples werewashed twice with staining buffer (PBS, 0.1% sodium azide, 2% fetalbovine serum), reconstituted in 500 μl of staining buffer and analyzedby a FACScan flow cytometer (Becton Dickinson). The fluorescence wasdisplayed as a single parameter histogram on a linear scale.

Measurement of P-selectin levels on surface of washed platelets obtainedfrom blood of mature individuals showed that approximately 50% of washedplatelets (resting platelets) tested positive for the presence ofP-selectin. Sixty-eight percent of the unwashed platelets obtained fromblood of an elderly individual tested positive for P-selectin. Whenwhole blood form this individual was supplemented with 10 μM finalconcentration of DHEA prior to fractionation of the platelets and thentest, only 12% of the platelets stained positive for P-selectin. Thisdown-regulation of P-selectin by DHEA was accompanied by a 40% reductionin thrombin activated platelet aggregation. When this latter individualwas placed on a supplemental therapy with DHEAS and the plateletsfractioned from blood drawn during the supplemental therapy with DHEAS,the platelets were refractory to exogenous DHEA when activated with thesame amount of thrombin as activated prior to the therapy. Thus, theobserved down-regulation of P-selectin on the surface of platelets fromelderly individuals by DHEA was accompanied by a prevention ofthrombin-stimulated aggregation of these platelets by DHEA.

When DHEAS is used in place of DHEA at 1.5 times the DHEA dose, similarresults are obtained. Similar results are obtained for the DHEAderivatives described above.

EXAMPLE 7 Effect of DHEA on Expression of P-Selectin by EndothelialCells

Non-virally transformed Human Dermal Microsvascular Endothelial cellswere cultured using conventional techniques. Cells in passage number 2were put on cover slips covered with attachment factor, and were grownin serum free system without phebol red until they became confluent.Groups of cells were incubated with vehicle alone or with 1 μM, 10 μM,25 μM, 50 μM or 100 μM DHEA at 37° C. for 10 minutes. The cells werethen activated with 10⁻⁵ M histamine or with Dulbecco's phosphate buffersaline (dPBS) at 37° C. for 5 minutes.

The cells were then examined by indirect immunostaining/fluorescencemicroscopy. Briefly the cells were first washed 2-3 times in dPBScontaining 1% bovine serum albumin (BSA), 1-2 minutes per wash. Thecells were then fixed in ice-cold methanol for 5-7 minutes and thenwashed 2-3 times in dPBS containing 1% BSA and 0.01% azide. The cellswere then incubated with anti P-selectin antibody at 4° C. in a humifiedchamber for 30 minutes. The cells were then washed 2-3 times in dPBScontaining 1% BSA at 4° C., 1-2 minutes per wash. The cells were then isincubated an anti anti-body linked to P-phycoerytherin at 4° C. for30-40 minutes, after which the cells were washed 2-3 times in dPBScontaining 1% BSA at 4° C., 1-2 minutes per wash. The slides are thenmounted and and P-selectin expression on endothelium is examined influorescence microscopy using rhodamine filterset.

Similary results are noted as seen for P-selectin expression inplatelets. Namely, DHEA at concentrations of 10 μM or greater preventedthe up-regulation of P-selectin expression normally observed onendothelium in response to histamine. The endothelium incubated withDHEA prior to histamine activation looked similar to the control,non-activated endothelium.

When DHEAS is used in place of DHEA, similar results are obtained.Similar results are obtained for the DHEA derivatives described above.

EXAMPLE 8 Effect of DHEAS on Hemorrhagic Shock

CF-1 mice, age 6-8 months, were anesthetized using methoxyflurothane andprepared for abdominal surgery. To maintain the required surgical levelof anesthesia, methoxyflurothane was used as needed in a nose coneapparatus. Each mouse was tested for the level of respiration, eye blinkresponse and response to a skin pinch to ensure a level of anesthesiaappropriate for surgery. The duration of abdominal surgery wasapproximately two hours, during which time 35-40% of the animal's bloodvolume is removed over a 30 minute period. The removal of blood in acontrolled manner simulates the effect of hemorrhagic shock. A slowintravenous infusion of the removed blood and a 2X volume ofresuscitation fluid (lactated Ringers solution) into a central vein wasmade. The resuscitation fluid was supplemented with either 2 mg DHEAS orthe excipient as a placebo. The peritoneum and overlying skin weresutured separately. Animals were maintained at 38°-39° C. until recoveryis complete. Under these conditions, most of the placebo-treated animalsdied within 24-48 hours. Four hours after surgery, a colony forming unit(CFU) assay for bacteria was performed and malondialdehyde in liver wasassayed using conventional techniques. Briefly, mesenteric lymph nodes(MLN) were removed and cultured on blood agar plates and the number ofCFUs counted following culturing. The liver was removed and the amountmalondialdehyde was measured. The survival rate, CFUs andmalondialdehyde results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                                             CFU at 4 Hours                                                                            Malondialdehyde                                Treatment Survival Post Surgery in Liver in 4                                 Group at 48 Hours (10.sup.6 /MLN cells) Hours (mMol)                        ______________________________________                                        Sham      15/15      0.8         0.035                                          Vehicle-treated,  1/15 12,020 0.226                                           shock/resusciation                                                            DHEAS-treated, 13/15 7.14 0.076                                               shock/resusciation                                                          ______________________________________                                    

When DHEA is used in place of DHEAS, similar results are obtained.Similar results are obtained for the DHEA derivatives described above.

EXAMPLE 9 Effect of DHEA on Hypoxia-Induced Pulmonary Vasoconstriction

Isolated perfused ferret lungs are an established animal model to studysecondary pulmonary hypertension, and were used in this example. Inbrief, male ferrets were anesthetized i.p. with pentobarbital sodium andthe chest was opened. Stainless steel cannulae were secured in the leftatrium and pulmonary artery, and the pulmonary artery and the aorta wereligated. The lungs were perfused with a mixture of autologous blood andKrebs-Henseleit buffer in a circulating manner at a constant rate of 85ml/min. The perfusion circuit included a perfusate reservoir, a rollerperfusion pump, filter, and a heat exchanger. The perfusion system wasmade of tygon tubing used for connections and for passage through theperfusion pump. The temperture of the perfusate was kept between 37 and38° C., the pH was maintained at 7.35 to 7.40 by adding sodiumbicarbonate to the reservoir as needed. The venous reservoir was placedbelow the lowermost portion of the lung.

The lungs were ventilated with a hypoxic gas mixture of 5% CO₂, 4% O₂,and 91% N₂ via a tracheotomy with a Harvard animal respirator for 30minutes. The animals were ventilated with a tidal volume of 30 ml, at arate of 18 breaths/min. and with 2 cm H₂ O positive end-expiatorypressure. For measurements, pulmonary arterial, left atrial and trachealpressures were monitored using Gould Statha P231D pressure transducersconnected to the inflow circulation and recorded on a Grass polygraph.After 30 minutes of ventilation with hypoxic gas mixture, DHEA in a dosebetween 8-12 mg/kg body weight was added to reservoir, and perfusate wasallowed to perfuse ferret lungs for 1.5 hours. A sudden drop to baselinelevel in pulmonary artery pressure was obserted upon DHEA delivery.Pulmonary artery pressure remained at basal level until the end of theexperiment, i.e., a total of two hours. These results demonstrate thevasodilatory effect of DHEA in pulmonary circulation constricted inresponse to hypoxia. DHEA treatment lowered pulmonary pressurecompletely to normal, and this lowering of pressure was sustained. Whencompared with nitric oxide (a therapeutic agent conventionally used) inthe same model, DHEA was more potent in reducing pulmonary arterypressure. The effect of nitric acid lasted for only minutes, whereas theeffect of DHEA lasted for at least two hours. Similar results areobtained for the DHEA derivatives described above.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent to theartisan that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

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What is claimed is:
 1. A method for preventing or reducing pulmonaryhypertension in a patient at risk of pulmonary hypertension whichcomprises administering to said patient an effective amount of adehydroepiandrosterone (DHEA) derivative having the general formulas Ior II or their pharmaceutically acceptable salts ##STR2## wherein R¹,R², R³, R⁴, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴ and R¹⁹ areindependently H, --OH, halogen, C₁₋₁₀ alkyl or C₁₋₁₀ alkoxy;R⁵ is H,--OH, halogen, C₁₋₁₀ alkyl, C₁₋₁₀ alkoxy or OSO₂ R²⁰ ; R¹⁵ is(1) H,halogen, C₁₋₁₀ alkyl or C₁₋₁₀ alkoxy when R¹⁶ is --C(O)OR²¹ or (2) H,halogen, OH or C₁₋₁₀ alkyl when R¹⁶ is H, halogen, OH or C₁₋₁₀ alkyl or(3) H, halogen, C₁₋₁₀ alkyl, C₁₋₁₀ alkenyl, C₁₋₁₀ alkynyl, formyl, C₁₋₁₀alkanoyl or epoxy when R¹⁶ is OH; or R¹⁵ and R¹⁶ taken together are ═O;R¹⁷ and R¹⁸ are independently(1) H, --OH, halogen, C₁₋₁₀ alkyl or C₁₋₁₀alkoxy when R¹⁶ is H, OH, C₁₋₁₀ alkyl or --C(O)OR²¹ or (2) H, (C₁₋₁₀alkyl)_(n) amino, (C₁₋₁₀ alkyl)_(n) amino-C₁₋₁₀ alkyl, C₁₋₁₀ alkoxy,hydroxy-C₁₋₁₀ alkyl, C₁₋₁₀ alkoxy-C₁₋₁₀ alkyl, (halogen)_(m) -C₁₋₁₀alkyl, C₁₋₁₀ alkanoyl, formyl, C₁₋₁₀ carbalkoxy or C₁₋₁₀ alkanoyloxywhen R¹⁵ and R¹⁶ taken together are ═O; or R¹⁷ and R¹⁸ taken togetherare ═O or taken together with the carbon to which they are attached forma 3-6 member ring containing 0 or 1 oxygen atoms; or R¹⁵ and R¹⁷ takentogether with the carbons to which they are attached form an epoxidering; R²⁰ is OH, pharmaceutically acceptable ester or pharmaceuticallyacceptable ether; R²¹ is H, (halogen)_(m) -C₁₋₁₀ alkyl or C₁₋₁₀ alkyl; nis 0, 1 or 2; and m is 1, 2 or 3,with the provisos that (a) R³ is not H,OH or halogen when R¹, R², R⁴, R⁶, R⁷, R⁹, R¹⁰, R¹², R¹³, R¹⁴, R¹⁷ andR¹⁹ are H and R is OH or C₁₋₁₀ alkoxy and R⁸ is H, OH or halogen and R¹¹is H or OH and R¹⁸ is H, halogen or methyl and R¹⁵ is H and R¹⁶ is OH;(b) R³ is not H, OH or halogen when R¹, R², R⁴, R⁶, R⁷, R⁹, R¹⁰, R¹²,R¹³, R¹⁴, R¹⁷ and R¹⁹ are H and R⁵ is OH or C₁₋₁₀ alkoxy and R⁸ is H, OHor halogen and R¹¹ is H or OH and R¹⁸ is H, halogen or methyl and R¹⁵and R¹⁶ taken together are ═O; (c) R⁵ is not H, halogen, C₁₋₁₀ alkoxy orOSO₂ R²⁰ when R₁, R₂, R₃, R⁴, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁴ and R¹⁷are H and R¹¹ is H, halogen, OH or C₁₋₁₀ alkoxy and R¹⁸ is H or halogenand R¹⁵ and R¹⁶ taken together are ═O; and (d) R⁵ is not H, halogen,C₁₋₁₀ alkoxy or OSO₂ R²⁰ when R¹, R², R³, R⁴, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹²,R¹³, R¹⁴ and R¹⁷ are H and R¹¹ is H, halogen, OH or C₁₋₁₀ alkoxy and R¹⁸is H or halogen and R¹⁵ is H and R¹⁶ is H, OH or halogen.
 2. The methodof claim 1, wherein R¹⁵ is H, halogen, C₁₋₁₀ alkyl or C₁₋₁₀ alkoxy andR¹⁶ is --C(O)OR²¹.
 3. The method of claim 1, wherein R¹⁵ is H, halogen,OH or C₁₋₁₀ alkyl and R¹⁶ is H, halogen, OH or C₁₋₁₀ alkyl.
 4. Themethod of claim 1, wherein R¹⁵ is H, halogen, C₁₋₁₀ alkyl, C₁₋₁₀alkenyl, C₁₋₁₀ alkynyl, formyl, C₁₋₁₀ alkanoyl or epoxy and R¹⁶ is OH.5. The method of claim 1, wherein R¹⁵ and R¹⁶ taken together are ═O. 6.The method of claim 1, wherein the compound is administeredintavenously.
 7. The method of claim 1, wherein the compound isadministered orally.
 8. The method of claim 1, wherein the compound isadministered in the amount of 1-1000 mg/kg.
 9. The method of claim 1,wherein the compound is administered in the amount of 2-200 mg/kg.